Integrated electro-analytical biosensor array

ABSTRACT

A biosensor pixel for measuring current that flows through the electrode surface in response to electrochemical interactions and a biosensor array architecture that includes such biosensor pixels. The biosensor pixel includes an electrode transducer configured to measure a current generated by electrochemical interactions occurring at a recognition layer placed directly on top of it in response to an electrical voltage placed across an electrode transducer-electrolyte interface. The biosensor pixel further includes a trans-impedance amplifier connected to the electrode transducer, where the trans-impedance amplifier is configured to convert the current into a voltage signal as the electrochemical interactions occur. Additionally, the biosensor pixel includes a 1-bit comparator coupled to the trans-impedance amplifier and a 1-bit digital-to-analog converter coupled to the 1-bit comparator, where the 1-bit digital-to-analog converter injects different levels of charge into an input of the trans-impedance amplifier at each cycle based on an output of the 1-bit comparator.

TECHNICAL FIELD

The present invention relates generally to biosensors andbioelectronics, and more particularly to a biosensor pixel configured tomeasure the current that flows through the electrode transducer surfacein response to and/or instantiated by electrochemical or biochemicalinteractions and a semiconductor-integrated biosensor array architecturethat includes a plurality of such biosensor pixels.

BACKGROUND

Biosensors are devices that use biochemical reactions to identify anddetect various molecules and biochemical analytes. Biosensors are widelyused in different life-science applications, ranging from environmentalmonitoring and basic life science research to Point-of-Care (PoC)in-vitro diagnostics. Biosensors are known to be very sensitive and alsoextremely versatile in terms of detection. They can efficiently detect asmall number of almost any type of analyte molecule or molecularstructure, once a proper recognition (capturing) molecule is identified.Example analytes that have been detected using biosensors include DNAand RNA strands, proteins, metabolites, toxins, micro-organisms, andeven explosives molecules.

All biosensors, independent of the analyte they are trying to detect,include two key building blocks. One is the molecular recognition layerattached to a solid-phase surface which is responsible for identifyingand/or interacting with and/or reacting with and/or capturing thespecific target analyte from the sample. The other is the sensorapparatus which detects and/or quantifies the interactions of therecognition layer with the analytes and provides a measurable output,generally in the form of an electrical signal. The molecular recognitionlayer typically comprises of carefully engineered and surface-assembledmolecules in addition to analyte-specific capturing molecules attachedto a solid-phase surface. Examples of such include spotted orsynthesized DNA oligonucleotides, aptamers, antigens or antibodiesattached to solid surfaces such as glass slides, micro-beads,electrodes, semiconductor materials, or dense polymers. Examples of asensor apparatus include optical-, MEMS- and/or electronics-basedtransducers connected to a low-noise electronic circuit.

So far, there have been many detection methods that have been adopted inbiosensor systems. A detection method is generally defined as thespecific type of physiochemical mechanism designed into the molecularrecognition layer, analytes, and the sample environment that make thecapturing of the specific target analytes detectable. The most widelyused detection methods are different types of optical (e.g.,fluorescence, bioluminescence) and electro-analytical (e.g.,potentiometric, amperometric, impedimetric). It is also customary toclassify biosensors based on their detection method (e.g., inbioluminescence-based biosensors, the interaction of the analyte andprobes results in a bioluminescence phenomenon which is detected by aspecific sensor with a transducer sensitive to bioluminescence signals).

One general class of biosensors which is relevant to the presentinvention is electro-analytical. The operating principle of suchbiosensors is based on measuring changes in the current, voltage orimpedance associated with biomolecular interactions taking place at arecognition layer at the interface of an electrode-electrolyteinterface. In these systems, the electrode typically acts as thesolid-phase on which the recognition layer is attached to the electrode.While electro-analytical methods are extremely versatile, they are morechallenging to implement compared to their optical counterparts. Themain reason is because of their transducer element (i.e.,electrode-electrolyte structure) which requires to be placed in intimateproximity of the recognition layer and capturing probes to efficientlysense analytes. In addition, creating large scale biosensor arrays,which are imperative in biotechnology high-throughput screeningapplications, are very difficult, as electrically accessing individualelectrodes within the array becomes the bottleneck.

SUMMARY

In one embodiment of the present invention, a biosensor pixel comprisesan electrode transducer with a recognition layer, where the electrodetransducer is configured to measure a current generated byelectrochemical interactions between an analyte and the recognitionlayer. The biosensor pixel further comprises a trans-impedance amplifierconnected to the electrode transducer, where the trans-impedanceamplifier is configured to convert the current into a voltage signal inreal-time as the electrochemical interactions occur. Additionally, thebiosensor pixel comprises a quantizer circuit coupled to thetrans-impedance amplifier with a differential input, where the quantizercircuit is configured to convert a value of the voltage signal into adigital value. Furthermore, the biosensor pixel comprises a chargeinjection circuit coupled to the quantizer circuit, where the chargeinjection circuit is configured to place a controllable current or a netcharge into an input of the trans-impedance amplifier. In addition, thebiosensor pixel comprises an in-pixel feedback network coupled to thequantizer circuit, where the feedback network comprises the chargeinjection circuit and where the feedback network is configured tocontrol an operation of the charge injection circuit based on values ofthe digital value.

In another embodiment of the present invention, a biosensor pixelcomprises an electrode transducer with a recognition layer, where theelectrode transducer is configured to measure a current generated byelectrochemical interactions between an analyte and the recognitionlayer. The biosensor pixel further comprises a trans-impedance amplifierconnected to the electrode transducer, where the trans-impedanceamplifier is configured to convert the current into a voltage signal inreal-time as the electrochemical interactions occur. Furthermore, thebiosensor pixel comprises a controlled voltage source coupled to apositive input of the trans-impedance amplifier to set a potential ofthe electrode transducer to a value of the controlled voltage source.Additionally, the biosensor pixel comprises a 1-bit comparator coupledto the trans-impedance amplifier. In addition, the biosensor pixelcomprises a 1-bit digital-to-analog converter coupled to the 1-bitcomparator, where the 1-bit digital-to-analog converter injectsdifferent levels of charge into an input of the trans-impedanceamplifier at each cycle based on an output of the 1-bit comparator.

In another embodiment of the present invention, a planar two-dimensional(2D) biosensor array architecture comprises a plurality of biosensorpixels assembled in rows and columns, where each of the plurality ofbiosensor pixels comprises an inert electrode transducer configured tosense a current generated by electrochemical interactions occurring atindividual recognition layer regions of every pixel in response todifferent electrical voltages being placed across an electrodetransducer-electrolyte interface for that pixel. Furthermore, each ofthe plurality of pixels comprises a trans-impedance amplifier connectedto the electrode transducer, where the trans-impedance amplifier isconfigured to convert the current into a voltage signal in real-time asthe electrochemical interactions occur. Additionally, each of theplurality of pixels comprises a controlled voltage source coupled to apositive input of the trans-impedance amplifier. Furthermore, each ofthe plurality of pixels comprises a 1-bit comparator coupled to thetrans-impedance amplifier. Additionally, each of the plurality of pixelscomprises a 1-bit digital-to-analog converter coupled to the 1-bitcomparator, where the 1-bit digital-to-analog converter injectsdifferent levels of charge into an input of the trans-impedanceamplifier at each cycle based on an output of the 1-bit comparator. Inaddition, the biosensor array architecture comprises row and columndecoders coupled to the plurality of pixels, wherein the row and columndecoders are configured to select individual pixels of the plurality ofpixels and access them one at a time.

The foregoing has outlined rather generally the features and technicaladvantages of one or more embodiments of the present invention in orderthat the detailed description of the present invention that follows maybe better understood. Additional features and advantages of the presentinvention will be described hereinafter which may form the subject ofthe claims of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention can be obtained when thefollowing detailed description is considered in conjunction with thefollowing drawings, in which:

FIG. 1 illustrates the basic block diagram of a pixel in accordance withan embodiment of the present invention;

FIG. 2A illustrates the cross-sections of a standard CMOS integratedcircuit in accordance with an embodiment of the present invention;

FIG. 2B illustrates a modified CMOS integrated circuit forelectro-analytical biosensing in accordance with an embodiment of thepresent invention;

FIGS. 3A-3C illustrate examples of the charge injection circuit of thepixel in accordance with an embodiment of the present invention;

FIG. 4 illustrates an alternative embodiment of the present invention ofa biosensor pixel;

FIG. 5 illustrates removing the idle tones in accordance with anembodiment of the present invention;

FIG. 6 illustrates an integrated biosensor array architecture inaccordance with an embodiment of the present invention;

FIG. 7 illustrates a biosensing pixel containing a working electrode, acounter electrode and a reference electrode in accordance with anembodiment of the present invention;

FIG. 8 is a micrograph of an implemented electro-analytical biosensorarray fabricated using a 0.18 μm CMOS process in accordance with anembodiment of the present invention;

FIG. 9 is a micrograph of an individual biosensor pixel in accordancewith an embodiment of the present invention;

FIG. 10 illustrates the transistor-level schematic of the detectioncircuitry within each biosensor pixel in accordance with an embodimentof the present invention;

FIGS. 11A-11C illustrate the measured noise power spectral density ofthe implemented system for different ΣΔ oversampling rates in accordancewith an embodiment of the present invention;

FIG. 12 illustrates the detailed structure of the fluidic cap which isplaced on the implanted biosensor array to allow the insertion andextraction of biological samples in accordance with an embodiment of thepresent invention; and

FIG. 13 illustrates a typical measured cyclic voltammetry waveform froman individual pixel when redox-based DNA detection is performed inaccordance with an embodiment of the present invention.

DETAILED DESCRIPTION

The principles of the preset invention relate to biosensors that useelectro-analytical detection methods for detection and quantification ofbio-molecules. There are two distinct components in the presentinvention that operate in concert to enable its functionality. One isthe molecular recognition layer component that identifies specificbio-molecules and/or bio-molecular structures from an aqueous sample andthe other is the electro-analytical sensor that translates suchmolecular identification events to a measurable output signal.

The present invention takes advantage of the recognition layer, attachedto a solid-phase, to identify bio-molecules and/or bio-molecularstructures that in the context of biosensors are generally referred toas “analytes.” The design, implementation, and fabrication of suchmolecular recognitions layers that typically consist of specificcapturing probes, such as DNA, RNA, or antibodies, attached to thesolid-phase through linker molecules are widely known in the art. Totranslate molecular recognition and capturing events into a measurablesignal, the present invention uses a current-based electro-analyticalsensor that is built using electronic circuits that are integrated in asemiconductor substrate.

In typical electro-analytical biosensors, changes in the current,voltage or the impedance, associated with the bio-molecular interactionsand reactions taking place are measured at an electrode-recognitionlayer interface which is in contact with the electrolyte. It is widelyknown in the art that such changes may occur when specific bio-moleculeanalytes, such as DNA, RNA, or peptides, herein referred to as thetarget molecules, interact with and/or are captured by the capturingprobes of the recognition layer during the biosensing process. Incertain cases, the target molecules can be chemically modified toinclude electro-active species, herein referred to as labels, toincrease the changes in the detectable signals. In such cases, uponsuccessful capturing, the labels generally get into the intimateproximity of the electrode and create unique electrochemicalinteractions and signals in response to an electrical voltage placedacross the electrode-recognition layer interface. In most cases, thelabels are a reduction-oxidation (redox) molecule that may or may notparticipate in a redox cycling process as a donor and/or an acceptor.Examples of such molecules include certain variants of organometalliccompounds, such as Ferrocene (Fe(C₅H₅)₂), or certain aromatic compounds,such as Methylene Blue (C₁₆H₁₈N₃SCl). Other examples of labels aredifferent redox enzymes, such as Glucose Oxidase or Horserdish Peroxide(HRP), which can create a highly electro-active molecule, such asHydrogen Peroxide (H₂O₂) using specific substrate molecules.

In the present invention, independent of using a label or not, thecurrent that flows through the electrode surface is specificallymeasured in real-time as an indicator of molecular interactions withinthe recognition layer. The target molecules captured at the recognitionlayer crate a unique current response when a specific electrical voltageis placed across the electrode-recognition layer interface. Such currentsignals during typical biosensing measurements are generally small(below 10 μA/mm² of electrode area and above 1fA/mm²) and typically varyslowly (below 10 kHz bandwidth); however, in certain embodiments of thepresent invention, the current is measured at higher frequencies. Hence,the principles of the present invention also implement electroniccircuits that enable high-performance current detection.

In embodiments of the present invention, the biosensor array is builtusing a complementary metal-oxide-semiconductor (CMOS) semiconductorsubstrate, in which the electro-analytical biosensor, including thecurrent sensor circuitry built using the active devices (e.g.,transistors and diodes) and passive devices (e.g., resistors, capacitorsand inductors), are built using the CMOS process. Furthermore, theelectrode transducers are also built using the metal layers of the CMOSback-end process.

In embodiments of the present invention the electro-analytical biosensorincludes a molecular recognition layer immobilized and attached to thesolid-phase surface of the electrode transducer integrated in a CMOSsubstrate. The capturing probe layer may include specific organicrecognition molecules, such as DNA strands or peptides which arechemically modified to attach directly to the surface of the electrodeor indirectly attach, through a linker molecule, to the surface of theelectrode.

In the description herein, methods are discussed to build “integratedelectro-analytical biosensor arrays,” which take advantage of electronicintegrated circuits (ICs) fabricated in a CMOS semiconductor substrate,as their sensing apparatus. In these systems, the biosensor array iscreated by placing recognition layers in intimate proximity of aCMOS-integrated electrode array that is connected to an integratedsensor circuitry embedded in the IC.

It is noted that biosensor arrays, including the systems describedherein, are essentially a plurality of densely packed biosensors thatcan detect multiple target molecules in parallel from a sample inreal-time. Individual sensors within the biosensor array are hereinreferred to as the “pixel.” In the context of the present invention,these pixels consist of an electrode transducer that contains therecognition layer and the dedicated integrated circuitry that performscurrent sensing, signal quantization and signal enhancement and/orsignal processing.

Electro-Analysis Pixel Architecture

The integrated electro-analytical biosensor array, discussed herein,consists of a plurality of independent pixels densely packed in asemiconductor substrate fabricated using processes, such as CMOS. Thenumber of pixels is typically greater than 10 and less than 10⁶.Furthermore, individual pixels may have distinct addressable recognitionlayers consisting of a specific capturing probe. Referring now to FIG. 1, FIG. 1 illustrates the basic block diagram of the pixel 100 inaccordance with an embodiment of the present invention. Pixel 100includes:

I. An electrode transducer 101 with a recognition layer 102 and acounter electrode 103 in the solution (electrolyte) 104, which can sensethe current that passes through its interface 105 that is generated bythe electrochemical interactions between the analyte 106 and recognitionlayer 102 that is denoted by I_(E);

II. A trans-impedance amplifier (TIA) 107, which converts I_(E) into avoltage signal while maintaining the potential of the voltage to V_(E).In certain embodiments of the present invention, a capacitancetrans-impedance amplifier circuit (CTIA) is used, which converts aninput current signal to a voltage by integrating the current onto itsfeedback capacitor, C_(F), using an operational amplifier (op-amp 108);

III. A quantizer circuit 109, which converts the analog output voltagevalue of TIA (or CTIA) 107 into a discrete and digital value D_(OUT);

IV. A charge injection circuit 110, which can insert or extract acontrollable current or net charge (ΔQ) into or out of the input of TIA(or CTIA) 107; and

V. A feedback network 111 which controls the operation of the chargeinjection circuit in real-time based on the values of D_(OUT).

Electrode Transducer 101 (FIG. 1 )

Most ICs today are built using semiconductor very large scaleintegration (VLSI) micro-fabrication processes. The CMOS fabricationprocess is currently the “flagship” process of VLSI micro-fabricationprocesses and the majority of ICs (both analog and digital) are builtusing this particular technology. In a typical CMOS process, activedevices (including MOS transistors) are fabricated in a planar siliconsubstrate on a wafer level; while interconnects (i.e., wirings) arebuilt using aluminum (and occasionally copper) metal layers embedded ina thin dielectric layer on top of the silicon substrate. One advantageof CMOS processes for electro-analysis, besides the capability tointegrate electronic circuit components, is that the metal layer can beused to create an electrode transducer. For example, this can be done bycreating an opening in the passivation layer that covers the top metallayer of a standard CMOS process as shown in FIGS. 2A-2B.

FIG. 2A illustrates the cross-sections of a standard CMOS integratedcircuit 201 in accordance with an embodiment of the present inventionand FIG. 2B illustrates a modified CMOS integrated circuit 202 forelectro-analytical biosensing in accordance with an embodiment of thepresent invention. Referring to FIGS. 2A and 2B, CMOS integrated circuit201 and modified CMOS integrated circuit 202 both include a siliconsubstrate 203, vias 204, aluminum interconnects 205 and a passivationlayer 206. Such components are well known in the art and will not bediscussed herein for the sake of brevity.

FIG. 2A further illustrates that while creating an opening 207 in thepassivation layer that covers the top metal layer is a simple andstraightforward approach, it is not the optimal solution forelectro-analytical biosensing as aluminum is not considered to be anoptimal metal for creating biosensing electrode transducers. This ismainly due to its susceptibility to corrosion when exposed to biologicalbuffers that are effectively highly conductive electrolytes. To addressthis problem, as shown in FIG. 2B, one can deposit or grow layers ofother materials on top of the aluminum surface and change it into arobust and bio- and electro-analytical-compatible electrode 208. Someexample bio-and electro-analytical compatible materials that can beplaced using various thin-film depositions methods on top of aluminumare noble metals, such as gold (Au) or platinum (Pt), or oxides, such asalumina, SiO₂, TiO₂ and HfO₂. The dimensions of such CMOS-integratedelectrodes 202 can vary from 0.2 μm×0.2 μm to 100 μm×100 μm and theintegrated biosensor array can have as low as 10 such electrodes to asmany as 10⁶. Also, the electrodes may have different shapes (e.g.,square, rectangular or circular).

It is deemed to be important to mention herein that the transducerelectrode of the present invention is effectively identical to theworking electrode that is used in conventional electro-analysis methodsincluding amperometry, voltammetry, and impedance spectroscopy.Typically, a three-electrode setup is used in conventionalelectro-analysis, where a reference electrode and a counter electrodeare required to be placed in the electrolyte in addition to the workingelectrode In the present invention, while the transducer electrode(i.e., working electrode) is incorporated in individual pixels, it isnot necessary to include a dedicated counter or reference electrode inevery pixel, as one can share the counter and the reference electrodesin the biosensor array systems without affecting the operation ofpixels. In other words, a plurality of the biosensing pixels in thearray may have a single reference and/or counter electrode. Suchreference and counter electrodes may or may not be integrated in CMOS asthe transducer electrode (working electrode) is created.

It is noted that there are many different methods to create recognitionlayers on top of the electrode. Such methods are widely known in thefield of electrochemistry and electro-analysis. Referring to FIG. 2B, inthe present invention, the electrode transducer includes a recognitionlayer that includes molecular capturing probes 209 immobilized andattached to the surface of electrode 208. Some example capturing probes209 are single-strand DNA, RNA strands, aptamers, proteins, orantibodies which are chemically modified to attach directly to thesolid-phase surface of electrode 208 or indirectly being immobilized,generally through a linker molecule 210, such as carbon chains,polymers, or sugars that are attached to the solid-phase surface ofelectrode 208.

Referring to FIGS. 1 and 2B in combination, it is noted that the currentgenerated in electro-analytical biosensors, denoted by I_(E), isbi-directional, meaning that the current can go into or come out oftransducer electrode 101. The directionality of the I_(E) depends on theapplied potential to the electrode, V_(E), the molecules involved in thesignal transduction process, including capturing probe 209 and targetanalyte 106, and the label (if any). Typically, V_(E) is a fixed DCpotential and is established by means of a negative feedback of TIA 107.However, in preferred embodiments, V_(E) can be a time-varying potentialsignal having a sine, square, saw-tooth waveform or a combination of theforegoing.

Capacitive Trans-Impedance Amplifier (CTIA) 107 (FIG. 1 )

Referring to FIG. 1 , CTIA 107 consists of opamp 108, negative feedbackcapacitor C_(F), a reset switch 112 across C_(F), and the voltage sourceV_(E) which is connected to the positive terminal of operationalamplifier 108 (see FIG. 1 ). In this system, if reset switch 112 isactivated at t<0 and released at t=0, then the output of CTIA 107,denoted by V_(OUT), at the end of measurement time, T_(S), becomes

$\begin{matrix}{{V_{OUT}\left( T_{S} \right)} = {{\frac{1}{C_{f}}{\int_{0}^{T_{S}}{{I_{E}(t)}{dt}}}} + V_{E}}} & \left( {{EQ}1} \right)\end{matrix}$

which for a constant I_(E) during T_(S), can be simplified to

$\begin{matrix}{{V_{OUT}\left( T_{S} \right)} = {\frac{I_{E}T_{S}}{C_{f}} + V_{E}}} & \left( {{EQ}2} \right)\end{matrix}$

One important characteristic of CTIA 107 is that the voltage at itsinput, which is effectively the electrode transducer voltage, followsV_(E). This is particularly useful when CTIA 107 is connected toelectrode transducer 101 by ensuring that the voltage applied to theelectrode is set to a controllable V_(E) and hence voltage across theelectrode-recognition layer 105 becomes tunable during the operation bysimply changing V_(E). As evident in (EQ 1) and (EQ 2), this has littleeffect on measuring I_(E) since changing V_(E) only adds a known offsetto the measured V_(OUT).

Quantizer Circuit 109 (FIG. 1 )

Referring to FIG. 1 , quantizer circuit 109 compares the output voltageat the end of the measurement time, V_(OUT)(T_(S)), to a single or aplurality of reference voltages in order to convert the V_(OUT)(T_(S))into a digital signal represented by D_(OUT). In the simplest case,quantizer 109 can be a 1-bit clocked (dynamic) comparator in whichV_(OUT) is compared to a fixed DC voltage, V_(C), at t=T_(S). In themore complicated case, quantizer 109 can be a multi-bitAnalog-to-Digital Converter (ADC), which is widely known in the art.

Charge Injection Circuit 110 (FIG. 1 )

Referring to FIG. 1 , charge injection circuit 110 performs the task ofinjecting a controlled current or net charge into the input node of CTIA107 during each measurement. There are multiple IC techniques toimplement this functionality. Some examples are:

A controlled-amplitude and adjustable current, I_(CAL), is directlyadded to or subtracted from the input of CTIA 107 (and integration ontoC_(F)). In this case, if I_(CAL) is remains unchanged during T_(S), thenthe net added or subtracted charge, dented by ΔQ, becomes equal toI_(CAL)×T_(S). FIG. 3A illustrates an example implementation of thiswhere two current sources (top PMOS and bottom NMOS) can addI_(CAL)=I_(ADD) or subtract I_(CAL)=I_(SUB) by enabling ADD or SUBdigital signals, respectively, in accordance with an embodiment of thepresent invention.

II. A fixed-amplitude current pulse, I_(S), with a controllable widthT_(CAL)(T_(CAL)<T_(S)), is used to add or subtract ΔQ=I_(S)×T_(CAL) fromthe input of CTIA 107. FIG. 3B illustrates an example implementation ofthis where clock Φ connects I_(CAL) onto the input of CTIA 107 forT_(CAL) second during T_(S) in accordance with an embodiment of thepresent invention.

III. A capacitor C_(S) is first charged to an adjustable referencevoltages, V_(CAL), and subsequently its stored charge ΔQ=C_(S)×V_(CAL)is added or subtracted from the input of CTIA 107 (and hence C_(F)).FIG. 3C illustrates an example switch-level implementation of this whereby toggling signal Φ, ΔQ is injected into the input of CTIA 107 inaccordance with an embodiment of the present invention.

Feedback Network 111 (FIG. 1 )

Referring to FIG. 1 , feedback network 111 effectively combines D_(OUT)with charge injection circuit 110. The goal of this block is todetermine and inject ΔQ within each measurement cycle based on D_(OUT)of the previous cycles. Feedback network 111 in the context of thepresent invention is typically implemented using digital circuits, whereD_(OUT) is first applied to a digital filter and/or gain blockscomprised of logic gates, adders, shift registers, and multipliers, andsubsequently, the result is used to control the functionality of chargeinjection circuits 110 previously discussed. In typical cases, theoutput of network 111 is a plurality of digital signals that controlsthe duration (e.g., T_(CAL)) and/or amplitude and/or frequency and/ordirectionality of injected charges.

Background Signal and Background Subtraction

Referring to FIG. 1 , I_(E) that passes through the electrode generallyconsist of two parts. One part is the current which is a function of theanalyte specific interactions with recognition layer 102 and the otheris the “background” signal which is independent of biosensing. Thebackground current is generally considered to be non-informative and itis preferred to be subtracted from the signal prior to detection. Inembodiments of the present invention, this current is subtracted byusing charge injection circuitry 110 at every cycle. In embodiments ofthe present invention, the subtracted charge is independent of D_(OUT)and feedback network 111.

Embodiment of the Pixel

FIG. 4 illustrates an alternative embodiment of the present invention ofa biosensor pixel 400 in accordance with an embodiment of the presentinvention. Referring to FIG. 4 , one pixel embodiment of the presentinvention is to implement a sigma-delta (Σ-Δ) modulator to measure I_(E)generated by electrochemical reactions at electrode transducer 101. Thisspecific circuit architecture offers the advantage of noise shapingcapabilities of Σ-Δ modulators, thereby improving the currentmeasurement dynamic range. In this pixel architecture, the feedbacknetwork forms a Digital-to-Analog Converter (DAC) 401 which subtracts ΔQthat represents D_(OUT) in each cycle (i.e., ever T_(S) seconds), whileCTIA 107 acts as the integrator of the Σ-Δ modulator as illustrated inthe biosensor pixel 400 of FIG. 4 .

In an example embodiment, a 1-bit quantizer 402 is used such thatD_(OUT)=0 for V_(OUT)(nT_(S))<V_(C), and D_(OUT)=1 forV_(OUT)(nT_(S))>V_(C), where n is an integer number indicating the cyclenumber. Feedback network 401 then subtracts ΔQ₁ and AQ₂ (ΔQ₁>ΔQ₂) forD_(OUT)=1 and D_(OUT)=0, respectively, at the next cycle, as shown inthe biosensor pixel 400 of FIG. 4 . The D_(OUT) sequence can then bedigitally filtered and down-sampled using a decimation filter toestimate I_(E).

In one embodiment, D_(OUT) changes T_(CAL), the width of the currentpulse I_(S) which is introduced at the input of CTIA 107. Hence, bymaking use of pulse width modulation (i.e., different pulse widthsT_(CAL)(1), T_(CAL)(2) T_(CAL)(N)) for different quantized D_(OUT)values D₁, D₂ . . . . DN, it is possible to create the feedback DAC 401and enable the Σ-Δ operation.

In another embodiment, the capacitor C_(F) is charged to differentreference voltages V_(REF)(1), V_(REF)(2), . . . , and V_(REF)(N), basedon D_(OUT) and its charge is then injected into the input of CTIA 107.

One advantage of the Σ-Δ modulator described herein is that it can alsoaccommodate background current subtraction without requiring anyadditional circuitry. The approach to do this is to subtract a fixedcharge that represents the background signal using DAC 401 and add thecharge representing D_(OUT) on top of that.

It is important to note here that utilizing 1-bit quantizers offer lowercomplexity, when compared to multi-bit quantizers. However, 1-bit Σ-Δmodulators inherently suffer from idle tones, when the input is a DCsignal. It is widely known in the art that these idle tones occur due tothe deterministic nature of the quantization noise and generally appearas tones with frequencies proportional to the input DC amplitudeapplied. In the present invention, such a problem is solved by usingnoise dithering which is a widely implemented technique in the field. Inone implementation of dithering, one can add a white noise source 501 tothe DC voltage source 502 at the input of voltage comparator 402 (FIG. 4) as shown in FIG. 5 to randomize the quantization noise and remove theidle tones in accordance with an embodiment of the present invention.

In one embodiment of the present invention, pixel 100, 400 (FIGS. 1 and4 ) is built using a CMOS fabrication process.

In one embodiment of the present invention, recognition layer 102 (FIGS.1 and 4 ) and electrode-transducer 101 (FIGS. 1 and 4 ) is placed on topof the pixel circuitry.

In one embodiment of the present invention, capture probes 209 (FIG. 2B)within recognition layer 102 and/or target molecules 106 (FIGS. 1 and 4) are nucleic acid strands, such as DNA or RNA or aptamers.

In an alternative embodiment, capturing probes 209 within recognitionlayer 102 and/or target molecules 106 include amino acid chains (e.g.,small peptides, proteins, antibodies).

In one embodiment, target molecules 106 are chemically modified toinclude electro-active labels to enhance the detectable signals. Thelabels may be a reduction-oxidation (redox) molecule that may or may notparticipate in a redox cycling process as a donor and/or an acceptor.Such molecules and structures are widely known in the art. Examplesinclude certain variants of organometallic compounds, such as Ferrocene(Fe(C₅H₅)₂), or certain aromatic compounds, such as Methylene Blue(C₁₆H₁₈N₃SCl). Other examples of labels include redox different redoxenzymes, such as Glucose Oxidase or Horserdish Peroxide (HRP), which cancreate a highly reactive molecule, such as Hydrogen Peroxide (H₂O₂)using specific substrate molecules.

In summary, referring to FIGS. 1, 4 and 5 , one embodiment of thepresent invention for a pixel includes:

I. An electrode transducer 101 with recognition layer 102 connected toCTIA 107;

II. A CTIA circuit 107 with its positive input connected to thecontrolled voltage source;

III. A 1-bit comparator 402 with a noise dithered reference voltage 501;and

IV. A 1-bit DAC 401 (based on D_(OUT)) which can inject different levelsof charge into the input of CTIA 107 at each cycle.

Biosensor Array Architecture

FIG. 6 illustrates an integrated electro-analytical biosensor arrayarchitecture 600 in accordance with an embodiment of the presentinvention. Pixels 601 are assembled in rows and columns within array600. Individual pixels 601 are selected by using row and column decoders602, 603, respectively. When a specific row within array 600 is selectedby means of a row decoder 602, D_(OUT) of all the pixels 601 in theselected row are connected to the shared column bus 604 by means ofpass-transistors (e.g., transmission gate switches 605). This allows theoutputs of the particular rows of interest to be connected to the columnlevel circuitry 606. Hence, by changing the inputs of the row and columndecoders 602, 603, the output of pixels 601 can be scanned and readsequentially.

The circuitry within column level circuitry 606 can offer multiplefunctionalities. In one embodiment, it connects a selected output of aselected pixel 601 to the output of the IC using a column decoder 603.In other embodiments, it can perform additional tasks, such as digitalfiltering, digital decimation and storage.

Array 600 can also include an on-chip power management and voltagegeneration circuitry 607, which ensures that all the blocks receive therequired DC supply and reference voltages required to set the operationpoint of the devices in individual pixels 601. Array 600 can alsoinclude a clock and timing generation block to control the timing of thepulses which go through pixels 601.

Electro-Analysis Setup

Electro-analytical detection requires additional electrodes in thesolution, besides the transducer (working) electrode of each pixel.Referring to FIG. 7 , FIG. 7 illustrates a biosensing pixel 700containing a working electrode, a counter electrode and a referenceelectrode in accordance with an embodiment of the present invention. Theintegrated electrode of each pixel 700 serves as the working electrode701 while another electrode 103, generally referred to as the counterelectrode, is also present in electrolyte 104 (or the biological buffersolution) to close the electrical circuit and allow I_(E) to flow.Beside working and counter electrodes 701, 103, one may use a referenceelectrode 702 to measure the potential of electrolyte 104 at relevantcoordinates within electrolyte 104 and adjust V_(E) to compensate forunwanted spatial potential variations. It is important to note here thatthe use of reference electrode 702 is optional within the scope of thepresent invention. In one embodiment, counter electrode 103 can becreated using many different methods and it can also be shared among aplurality of pixels 601 (FIG. 6 ) within array 600 (FIG. 6 ). Forexample, a thin wire made from non-corroding noble metals (e.g., Pt orAu) can be placed in the solution as counter electrode 103.Alternatively, counter electrodes 103 can be integrated using the metallayers of a CMOS process in similar fashion to working electrodes 701and be placed in all pixels 601.

In one embodiment, reference electrode 702 can be created using the samemethods as used in creating counter electrode 103 and may or may not beshared among pixels 601. In a specific example, large Ag/AgCl or Mg/MgClreference electrodes 702 can be used and shared among pixels 601.

Biosensing Setup

An electro-analytical biosensor array, such as array 600 (FIG. 6 ), canbe used in molecular detection assays, where the change in I_(E) ismeasured to determine the presence or the concentration of the analyteof interest. In the present invention, integrated biosensor arrays aredeveloped, in which capturing probe 209 (FIG. 2B) within recognitionlayer 102 (FIGS. 1, 4 and 7 ) is directly placed on top of andintegrated with the integrated circuit. This permits the development ofan integrated and compact detection platform, in which molecularrecognition and sensing are done using the same device.

In one embodiment of the present invention, electro-analytical biosensorarray 600 can be used in affinity-based detection of multiple targetmolecules from a single biological sample interfaced with and/orencapsulated on top of the biosensor array.

In an alternative embodiment of the present invention,electro-analytical biosensor array 600 can be used in affinity-baseddetection of multiple target molecules 106 (FIGS. 1 and 4 ) in a samplethat is flowing through the surface of biosensor array 600.

Example Embodiment

As an example embodiment, a fully-integrated electro-analyticalbiosensor array was fabricated using a Taiwan SemiconductorManufacturing Company (TSMC) 0.18 μm mixed-signal CMOS process thatcontains 6 metal layers for interconnects on top of its siliconsubstrate. FIG. 8 illustrates the micrograph of this 2.5 mm×2.5 mm IC800 in accordance with an embodiment of the present invention. Referringto FIG. 8 , chip 800 includes a 12×12 array 600 of biosensing pixels601, where each pixel 601 occupies a 120 μm×120 μm area as shown in FIG.8 .

Referring to FIG. 9 , FIG. 9 is a micrograph of an individual biosensorpixel 601 in accordance with an embodiment of the present invention.Each pixel 601 in the chip includes a working electrode 701 and a Σ-Δcurrent detection system 901 including CTIA 107 (FIG. 4 ), whichincludes operational amplifier 108 and capacitor C_(F), voltagecomparator 402 (FIG. 4 ) and the switches of 1-bit DAC 401 (FIG. 4 ).The reference and counter electrodes 702, 103 (FIG. 7 ) (not shown inFIG. 9 ) in this system are shared among all pixels 601 and reside inthe reaction chamber containing the analyses on the top pixel array,making the chip usable for electro-analysis. Furthermore, as illustratedin FIG. 9 , each pixel 601 includes a top metal layer 902 surroundingworking electrode 701.

FIG. 10 illustrates the transistor-level schematic of the detectioncircuitry within each biosensor pixel 601 (FIG. 6 ) in accordance withan embodiment of the present invention. Specifically, FIG. 10illustrates the circuit-level schematic of the implementedelectro-analytical biosensing pixels intended to measure I_(E) thatflows through working electrode 701 (FIG. 7 ) of each pixel 601.Referring to FIG. 10 , in conjunction with FIG. 1 , as shown, CTIA 107comprises operational amplifier 108 and programmable capacitive feedbacknetwork, which can be set to have values C_(F1) or C_(F2)+C_(F2), basedon the logical value of the GAIN signal. The 1-bit quantizer 109 isbuilt using a CMOS clocked (dynamic) comparator with OUT and OUTBdifferential outputs that control, through direct feedback, the value ofthe injected charge using the switch network of charge injection block110. The digital output of this pixel 601 (such as pixel 100) isconnected to the shared column bus using the in-pixel decoder circuitry1001.

FIGS. 11A-11C illustrate the measured noise power spectral density ofthe implemented system for different ΣΔ oversampling rates (FIG. 11Acorresponds to the oversampling rate of 1/T_(s) equal to 1 MHz, FIG. 11Bcorresponds to the oversampling rate of 1/T_(s) equal to 500 kHz andFIG. 11C corresponds to the oversampling rate of 1/T_(s) equal to 100kHz) in accordance with an embodiment of the present invention. Asevident in all cases, 1^(st)-order noise shaping occurs, but dependingon the oversampling rate, the useful bandwidth of the system decreasesas 1/T_(s) decreases. However, for a fixed detection bandwidth of DC to100 Hz, which was the specification for this implemented system, thenoise performance of the system improves as 1/T_(s) decreases andreaches the 1.5 pA r.m.s input-referred noise at 1/T_(s)=100 kHz.

FIG. 12 illustrates a fluidic cap 1201 which is placed on the implantedbiosensor array 600 to allow the insertion and extraction of biologicalsamples in accordance with an embodiment of the present invention. Inone embodiment, the function of fluidic cap 1201 is to enable a flowthrough a fluidic system 1202 to introduce the sample of biosensor array600 (FIG. 6 ), and if necessary, extract it through an inlet 1203 andoutlet 1204 while allowing electrical access to the chip 600 throughwire-bonds connected to the CMOS IC pads at its periphery. In oneembodiment, the volume for the reaction chamber within this cap isapproximately 100 μl.

FIG. 13 illustrates a typical measured cyclic voltammetry waveform froman individual pixel when redox-based DNA detection is performed inaccordance with an embodiment of the present invention. In particular,FIG. 13 illustrates a typical example of how the system can detectspecific DNA strands (as target analytes) in accordance with embodimentsof the present invention.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

1. A biosensor pixel, comprising: an electrode transducer with arecognition layer, wherein said electrode transducer is configured tomeasure a current generated by electrochemical interactions between ananalyte and said recognition layer; a trans-impedance amplifierconnected to said electrode transducer, wherein said trans-impedanceamplifier is configured to convert said current into a voltage signal inreal-time as said electrochemical interactions occur; a quantizercircuit coupled to said trans-impedance amplifier with a differentialinput, wherein said quantizer circuit is configured to convert a valueof said voltage signal into a digital value; a charge injection circuitcoupled to said quantizer circuit, wherein said charge injection circuitis configured to place a controllable current or a net charge into aninput of said trans-impedance amplifier; and an in-pixel feedbacknetwork coupled to said quantizer circuit, wherein said feedback networkcomprises said charge injection circuit, wherein said feedback networkis configured to control an operation of said charge injection circuitbased on values of said digital value.
 2. The biosensor pixel as recitedin claim 1, wherein said recognition layer comprises capture probes. 3.The biosensor pixel as recited in claim 2, wherein said capture probescomprise nucleic acid strands.
 4. The biosensor pixel as recited inclaim 2, wherein said capture probes comprise amino acid chains.
 5. Thebiosensor pixel as recited in claim 2, wherein a layer of said captureprobes comprises organic recognition molecules.
 6. The biosensor pixelas recited in claim 5, wherein said organic recognition moleculescomprise DNA strands which are chemically modified to attach directly toa surface of said electrode transducer.
 7. The biosensor pixel asrecited in claim 5, wherein said organic recognition molecules compriseDNA strands which are chemically modified to attach indirectly to asurface of said electrode transducer through a linker molecule.
 8. Thebiosensor pixel as recited in claim 1, wherein said analyte compriseselectro-active labels.
 9. The biosensor pixel as recited in claim 8,wherein said labels comprise reduction-oxidation (redox) molecules. 10.A biosensor pixel, comprising: an electrode transducer with arecognition layer, wherein said electrode transducer is configured tomeasure a current generated by electrochemical interactions between ananalyte and said recognition layer; a trans-impedance amplifierconnected to said electrode transducer, wherein said trans-impedanceamplifier is configured to convert said current into a voltage signal inreal-time as said electrochemical interactions occur; a controlledvoltage source coupled to a positive input of said trans-impedanceamplifier to set a potential of said electrode transducer to a value ofsaid controlled voltage source; a 1-bit comparator coupled to saidtrans-impedance amplifier; and a 1-bit digital-to-analog convertercoupled to said 1-bit comparator, wherein said 1-bit digital-to-analogconverter injects different levels of charge into an input of saidtrans-impedance amplifier at each cycle based on an output of said 1-bitcomparator; wherein said recognition layer comprises capture probes. 11.The biosensor pixel as recited in claim 10, wherein said capture probescomprise amino acid chains.
 12. The biosensor pixel as recited in claim10, wherein a layer of said capture probes comprises organic recognitionmolecules.
 13. The biosensor pixel as recited in claim 12, wherein saidorganic recognition molecules comprise DNA strands which are chemicallymodified to attach directly to a surface of said electrode transducer.14. The biosensor pixel as recited in claim 12, wherein said organicrecognition molecules comprise DNA strands which are chemically modifiedto attach indirectly to a surface of said electrode transducer through alinker molecule.
 15. A planar two-dimensional biosensor arrayarchitecture, comprising: a plurality of biosensor pixels assembled inrows and columns, wherein each of said plurality of biosensor pixelscomprises: an inert electrode transducer configured to sense a currentgenerated by electrochemical interactions occurring at individualrecognition layer regions of every pixel in response to differentelectrical voltages being placed across an electrodetransducer-electrolyte interface for that pixel; a trans-impedanceamplifier connected to said electrode transducer, wherein saidtrans-impedance amplifier is configured to convert said current into avoltage signal in real-time as said electrochemical interactions occur;a controlled voltage source coupled to a positive input of saidtrans-impedance amplifier; a 1-bit comparator coupled to saidtrans-impedance amplifier; and a 1-bit digital-to-analog convertercoupled to said 1-bit comparator, wherein said 1-bit digital-to-analogconverter injects different levels of charge into an input of saidtrans-impedance amplifier at each cycle based on an output of said 1-bitcomparator; and row and column decoders coupled to said plurality ofbiosensor pixels, wherein said row and column decoders are configured toselect individual pixels of said plurality of biosensor pixels andaccess them one at a time; wherein a recognition layer of a biosensorpixel of said plurality of biosensor pixels comprises capture probes.16. The biosensor array architecture as recited in claim 15, whereinsaid capture probes comprise amino acid chains.
 17. The biosensor arrayarchitecture as recited in claim 15, wherein a layer of said captureprobes comprises organic recognition molecules.
 18. The biosensor arrayarchitecture as recited in claim 17, wherein said organic recognitionmolecules comprise DNA strands which are chemically modified to attachdirectly to a surface of said electrode transducer.
 19. The biosensorarray architecture as recited in claim 17, wherein said organicrecognition molecules comprise DNA strands which are chemically modifiedto attach indirectly to a surface of said electrode transducer through alinker molecule.